Summary Age-related defects in human immune function have been most studied in the context of influenza vaccination and infection, or other settings where an individual's history of prior antigenic exposures and adaptive immune memory complicates analysis. In contrast, both young and elderly individuals are also able to form new primary immune responses following novel antigen exposures. Vaccine-mediated protection of communities against emergent infectious diseases such as Ebola and Zika viruses will rely on new primary adaptive immune responses across age categories. We will comprehensively analyze the adaptive immune responses in young adult and elderly subjects to three different kinds of primary vaccinations administered by parenteral or oral routes: the Hepatitis A (HAV) inactivated and aluminum salt-adjuvanted viral vaccine, and two vaccines for Typhoid (injected polysaccharide or oral attenuated bacterial vaccine). Importantly, we will study these primary immune responses in a unique clinical cohort: the longitudinal Stanford-Ellison cohort of young adult (20-35 years) and elderly (60-95 years) subjects whose yearly influenza vaccine responses have been studied extensively for the past nine years, and will continue to be studied during the grant period so that explicit comparison of primary and secondary responses can be made in the same individuals. We will use novel single B cell and T cell antigen receptor repertoire analysis and transcriptional phenotyping methods, and single cell monoclonal antibody expression and characterization, to define the B cell and T cell clones that respond to each primary vaccination. In a subset of subjects, we will also obtain bone marrow aspirates to analyze the bone marrow plasma cell populations, and identify which clones from the acute vaccine responses in the blood contribute to the bone marrow plasma cell pool versus the memory B cell pool. In addition, gut microbiota data will be collected to evaluate for potential interactions between vaccines and microbiota. The impact of this Project will stem from a combination of opportunities for comprehensive study of primary and secondary adaptive immune responses and the effects of aging on human immune systems: 1) a uniquely well-characterized longitudinal cohort of young and elderly subjects in whom clinically-relevant primary vaccine responses can be studied in parallel with secondary responses to influenza vaccination, 2) single-cell characterization of vaccine-specific B cell and T cell receptor repertoires and phenotypes in combination with a panel of standard immunological assays, 3) tracking of vaccine-stimulated B cell clones from the acute vaccine response in the blood, to residence in the bone marrow plasma cell pool, and 4) integration with microbiome data and other significant clinical data.